Modern Pathology (2010) 23, 1261–1268 & 2010 USCAP, Inc. All rights reserved 0893-3952/10 $32.00 1261 Prognostic significance of HER2 expression in neuroblastic tumors Ewa Izycka-Swieszewska1, Agnieszka Wozniak2,3, Jacek Kot4, Wieslawa Grajkowska5, Anna Balcerska6, Danuta Perek7, Bozena Dembowska-Baginska7, Teresa Klepacka8 and Elzbieta Drozynska6 1Department of Pathomorphology, Medical University of Gdansk, Gdansk, Poland; 2Department of Biology and Genetics, Medical University of Gdansk, Gdansk, Poland; 3Department of General Medical Oncology, Laboratory of Experimental Oncology, University Hospitals Catholic University Leuven, Leuven, Belgium; 4Department of Hyperbaric Medicine and Sea Rescue, Medical University of Gdansk, Gdynia, Poland; 5Department of Pathology Children’s Health Memorial Institute, Warsaw, Poland; 6Department of Pediatrics, Hematology, Oncology and Endocrinology, Medical University of Gdansk, Gdansk, Poland; 7Department of Oncology, Children’s Health Memorial Institute, Warsaw, Poland and 8Department of Pathology, Institute of Mother and Child, Warsaw, Poland HER2 is essential for normal embryonic development and has a critical function in oncogenesis and progression of some types of cancer. Neuroblastic tumors create a heterogenous group of pediatric embryonal tumors of sympathoadrenal lineage. The biological and prognostic function of HER2 in these tumors is not well established. In this study, we evaluated the status of HER2, its prognostic significance, and clinicopathological correlations in series of 79 untreated neuroblastoma. The immunohistochemical assessment of HER2 and Ki-67 (proliferation index) as well as HER2 copy number status were performed on tissue microarrays. HER2 expression characterized 63 tumors, including 34 with low and 29 with high level, showing either membranous or mixed membranous–cytoplasmic pattern. Sixteen cases were HER2 immunonegative. The pattern of immunolabeling depended on the maturity of neuroblastic cells, being the most intense in differentiating neuroblasts. None of the tumors revealed HER2 amplification. In the examined group, 20% of patients died of disease from 4 to 107 months (median 18) from the diagnosis, and the survivors were followed up for 14–149 months (median 59). Patients’ age, stage of disease, tumor location, mitosis/karyorrhexis index (MKI), and presence of HER2 expression were statistically significantly related to survival probability as detected by the Cox proportional hazard model. In the univariate analysis, Kaplan–Meier curves revealed significantly poorer outcome of HER2 negative than HER2-positive tumors (either low or high expression). The immunonegativity was associated with adverse clinicopathological parameters, including poor survival, metastatic stage of disease, un- or poorly differentiated histology, high MKI, and higher proliferation index. In conclusion, HER2 expression, not accompanied by gene amplification, is common in neuroblastic tumors. HER2 positivity seems to have a positive prognostic significance. HER2 expression with a variable pattern is a marker of the stage of neuroblastic cells differentiation. Modern Pathology (2010) 23, 1261–1268; doi:10.1038/modpathol.2010.115; published online 25 June 2010 Keywords: FISH; HER2 protein expression; HER2 status; neuroblastoma; patients’ survival; prognostic factor; proliferation index Neuroblastic tumors are the most frequent extra- cranial infantile solid tumors and common pediatric Correspondence: E Izycka-Swieszewska, MD, PhD, Department of neoplasms of the sympathetic ganglia and adrenal Pathomorphology, Medical University of Gdansk, Debinki medulla.1–3 They develop from the persistent popu- 7 Street, 80-211 Gdansk, Poland. lation of embryonal neural crest cells that would E-mail: [email protected] 1–3 Received 28 January 2010; revised and accepted 6 May 2010; have either differentiated or undergo apoptosis. published online 25 June 2010 Neuroblastic tumors are remarkable for their diverse www.modernpathology.org HER2 in neuroblastoma 1262 E Izycka-Swieszewska et al pathological picture concerning the level of neo- The results for HER2 expression and its biological plastic cells differentiation and Schwannian stroma function in neuroblastic tumors are sparse and amount as well as diverse ways of biological controversial. The aim of this study was to evaluate behavior ranging from spontaneous regression to the status of HER2 in our series of neuroblastoma the lethal metastatic spread.1–3 The new Interna- and to assess its prognostic significance and clinico- tional Neuroblastoma Risk Group classification pathological correlations. system uses relevant factors, including tumor stage, patients’ age, histologic category, grade of tumor differentiation, the status of MYCN gene and Materials and methods chromosome 11q status as well as DNA ploidy.4 A group of 79 patients with neuroblastic tumors Nowadays, the status of these factors is necessary for diagnosed and treated between 1996 and 2007 in the adjustment of therapeutic protocols ranging three pediatric oncological centers in Poland from the ‘wait and see’ strategy, throughout multi- (Department of Pediatrics, Hematology, Oncology modal surgery, adjuvant chemotherapy, and radio- and Endocrinology, Medical University of Gdansk; therapy up to megachemotherapy with bone marrow Department of Pediatric Oncology Children’s Health transplantation.3–5 About half of all cases are Memorial Institute; and Institute of Mother and classified as high-risk with long-term survival rates Child in Warsaw) was included in the study, based 40% despite intensive complex treatment.3,5 Bet- o on availability of tissue material. Clinical data ter understanding of signaling pathways involved in comprised patients’ age, primary tumor localization, neuroblastoma tumorigenesis and disease progres- stage of disease, and overall survival at the time of sion may allow to develop more effective therapies the last observation (March 2009). Molecular data based on individualized tumor predicitive biomar- included tumor cell ploidy and MYCN status. kers profile. More than 20 years ago, human epidermal growth factor receptor 2 (ERBB2, HER2) encoded by the Immunohistochemistry HER2 gene was first identified as a transforming gene in ethylnitrosourea-induced rat neuroblasto- The study was performed on the archival material of ma.6 This transmembrane glycoprotein—member of routinely processed tumor sections taken at the EGFRs tyrosine kinase receptors family mediates surgical biopsy or tumor resection before chemother- many cellular signaling pathways in developmental, apy. The hematoxylin and eosin-stained slides were physiological, and neoplastic processes.7–10 So far, reviewed by two independent pathologists (EIS and there has been no HER2 ligand identified and the WG) to verify the diagnosis, establish mitosis/ receptor acts on the way of homo- or heterodimeri- karyorrhexis index (MKI), and choose the repre- zation with the other EGFR family receptors.7,11 sentative sections for further evaluation. In the HER2 has a pivotal function in embryogenesis next step, the tissue microarrays (TMAs) were dictating the establishment of several cell lineages constructed using Tissue-Tek Quick-Ray Tissue through mesenchyme-epithelial-neuroectodermal Microarray System (Sakura Finetek USA) according inductive processes.8,9,12 It influences cellular to the manufacturer’s instructions. One or two 5 mm migration, differentiation, and interactions between core biopsies were obtained for each tumor and cells.8–10,12 The critical function of ERBB2 is mounted into recipient blocks. exemplified in murine erbb2 mutants, which The following antibodies were used for immuno- display an abnormal structure of heart, peripheral histochemical evaluation: C-terminal c-erbB-2 nervous system, and sympathetic ganglia, absence of (pc. rabbit, 1:50, DAKO, A 0485), external domain Schwann cells, and lack of functional neuro- of c-erbB-2 (mc. Novocastra 10A7), and Ki-67 (1:100, muscular junction.8,12 DAKO, F 0788), with appropriate positive and The tumorigenic potential of abnormally activated negative controls. A standard protocol with heat- HER2 is supported by many experimental mod- induced antigen retrieval in citrate buffer was els.7,11,13 HER2 upregulates cellular proliferation performed. Subsequently, the slides were incubated and migration by sustained activation of multiple with the primary antibody for 30 min in a room signaling pathways, including mitogen-activated temperature. Thereafter, visualization system En protein kinase and phosphoinositide-3-kinas/AKT Vision and DAB (DAKO) with hematoxylin staining cascades.7,9,11 Amplification of HER2 gene and were performed. overexpression of the encoded protein are asso- HER2 expression was analyzed with both c-erbB-2 ciated with a poor prognosis in several types of antibodies in neuroblastic cell component of tumors cancer.7,11,14 Based on that, selective HER2 inhibitors in a descriptive and semiquantitative method. Some have been successfully introduced in breast cancer cases contained surrounding suprarenal gland that and have been tested in other tumor types as the was used for the internal control. We also examined target therapy.7,11,14,15 On the other hand, in many five normal paraspinal ganglia (taken at the autopsy) tumors, HER2 expression does not influence the as well as two spontaneously aborted fetuses patients’ survival or even less frequently is a (10, 13th week of the pregnancy). In the quanti- favorable prognostic factor.13,16,17